Genomic and biochemical insights into the specificity of ETS transcription factors.

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5'-GGA(A/T)-3'. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.

Biophysical characterization of the ETV6 PNT domain polymerization interfaces.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Conformational Plasticity and DNA-Binding Specificity of the Eukaryotic Transcription Factor Pax5.

The eukaryotic transcription factor Pax5 has a DNA-binding Paired domain composed of two independent helical bundle subdomains joined by a flexible linker. Previously, we showed distinct biophysical properties of the N-terminal (NTD) and C-terminal (CTD) subdomains, with implications for how these two regions cooperate to distinguish nonspecific and cognate DNA sites [Perez-Borrajero, C., et al. (2016) J. Mol. Biol. 428, 2372-2391]. In this study, we combined experimental methods and molecular dynamics (MD) simulations to dissect the mechanisms underlying the functional differences between the Pax5 subdomains. Both subdomains showed a similar dependence of DNA-binding affinity on ionic strength. However, due to a greater contribution of non-ionic interactions, the NTD bound its cognate DNA half-site with an affinity approximately 10-fold higher than that of the CTD with its half-site. These interactions involve base-mediated contacts as evidenced by nuclear magnetic resonance spectroscopy-monitored chemical shift perturbations. Isothermal titration calorimetry revealed that favorable enthalpic and compensating unfavorable entropic changes were substantially larger for DNA binding by the NTD than by the CTD. Complementary MD simulations indicated that the DNA recognition helix H3 of the NTD is particularly flexible in the absence of DNA and undergoes the largest changes in conformational dynamics upon binding. Overall, these data suggest that the differences observed for the subdomains of Pax5 are due to the coupling of DNA binding with dampening of motions in the NTD required for specific base contacts. Thus, the conformational plasticity of the Pax5 Paired domain underpins the differing roles of its subdomains in association with nonspecific versus cognate DNA sites.

Ligand- and pH-Induced Structural Transition of Gypsy Moth Lymantria dispar Pheromone-Binding Protein 1 (LdisPBP1).

Pheromone-binding proteins (PBPs) are small, water-soluble proteins found in the lymph of pheromone-sensing hairs. PBPs are essential in modulating pheromone partitioning in the lymph and at pheromone receptors of olfactory sensory neurons. The function of a PBP is associated with its ability to structurally convert between two conformations. Although mechanistic details remain unclear, it has been proposed that the structural transition between these forms is affected by two factors: pH and the presence or absence of ligand. To better understand the PBP conformational transition, the structure of the gypsy moth (Lymantria dispar) LdisPBP1 was elucidated at pH 4.5 and 35 °C using nuclear magnetic resonance spectroscopy. In addition, the effects of sample pH and binding of the species' pheromone, (+)-disparlure, (7R,8S)-epoxy-2-methyloctadecane, and its enantiomer were monitored via 15N HSQC spectroscopy. LdisPBP1 in acidic conditions has seven helices, with its C-terminal residues forming the seventh helix within the pheromone-binding pocket and its N-terminal residues disordered. Under conditions where this conformation is made favorable, free LdisPBP1 would have limited ligand binding capacity due to the seventh helix occupying the internal binding pocket. Our findings suggest that even in the presence of 4-fold ligand at acidic pH, LdisPBP1 is only ∼60% in its pheromone-bound form. Furthermore, evidence of a different LdisPBP1 form is seen at higher pH, with the transition pH between 5.6 and 6.0. This suggests that LdisPBP1 at neutral pH exists as a mixture of at least two conformations. These findings have implications concerning the PBP ligand binding and release mechanism.

Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids.

Understanding the detailed mechanisms of enzyme-catalyzed hydrolysis of the glycosidic bond is fundamentally important, not only to the design of tailored cost-efficient, stable and specific catalysts but also to the development of specific glycosidase inhibitors as therapeutics. Retaining glycosidases employ two key carboxylic acid residues, typically glutamic acids, in a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. One Glu functions as a nucleophile while the other acts as a general acid/base. A significant part of enzymatic proficiency is attributed to a "perfect match" of the electrostatics provided by these key residues, a hypothesis that has been remarkably difficult to prove in model systems or in enzymes themselves. We experimentally probe this synergy by preparing synthetic variants of a model glycosidase Bacillus circulans ß-xylanase (Bcx) with the nucleophile Glu78 substituted by 4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity. These Bcx variants were semisynthesized by preparation of optically pure fluoroglutamic acid building blocks, incorporation into synthetic peptides, and ligation onto a truncated circular permutant of Bcx. By measuring the effect of altered electrostatics in the active site on enzyme kinetic constants, we show that lowering the nucleophile p Ka by two units shits the pH-dependent activity by one pH unit. Linear free energy correlations using substrates of varying leaving group ability indicate that by reducing nucleophilic catalysis the concerted mechanism of the enzyme is disrupted and shifted toward a dissociative pathway. Our study represents the first example of site-specific introduction of fluorinated glutamic acids into any protein. Furthermore, it provides unique insights into the synergy of nucleophilic and acid/base catalysis within an enzyme active site.

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor.

The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.

Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

Structural characterization of the C3 domain of cardiac myosin binding protein C and its hypertrophic cardiomyopathy-related R502W mutant.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

Structure-function analysis of a broad specificity Populus trichocarpa endo-ß-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products.

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/ß-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage ß(1→3)/ß(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins.

Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

pH-dependent random coil (1)H, (13)C, and (15)N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements.

The pK a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the (1)H, (13)C, and (15)N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain (15)N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy.

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

Investigating the structural dynamics of α-1,4-galactosyltransferase C from Neisseria meningitidis by nuclear magnetic resonance spectroscopy.

Neisseria meningitidis α-1,4-galactosyltransferase C (LgtC) is responsible for the transfer of α-galactose from donor UDP-galactose to the lipooligosaccharide terminal acceptor lactose. Crystal structures of its substrate analogue complexes have provided key insights into the galactosyl transfer mechanism, including a hypothesized need for active site mobility. Accordingly, we have used nuclear magnetic resonance spectroscopy to probe the structural dynamics of LgtC in its apo form and with bound substrate analogues. More than the expected number of signals were observed in the methyl-TROSY spectra of apo LgtC, indicating that the protein adopts multiple conformational states. Magnetization transfer experiments showed that the predominant states, termed "a" and "b", are in equilibrium on a time scale of seconds. Their relative populations change with temperature and mutations, and only the "b" state is competent for substrate binding. For both states, relaxation dispersion studies also revealed substantial millisecond time scale motions of isoleucine side chains within and distal to the active site. Although altered, these motions were still detected in LgtC with a noncovalently bound donor analogue. A mutant, LgtC-Q189E, which forms an unexpected glycosyl-enzyme intermediate via a residue (Asp190) distal from its active site, was also investigated. Apo LgtC-Q189E did not show any enhanced motions that might account for the dramatic structural change required for the galactosylation of Asp190, yet formation of a trapped glycosyl-enzyme intermediate substantially reduced its millisecond time scale conformational mobility. Although further studies are required to link the detected motions of LgtC with its enzymatic mechanism, this work clearly demonstrates the complex structural dynamics of a model glycosyltransferase.

Strategies for modulating the pH-dependent activity of a family 11 glycoside hydrolase.

The pH-dependent activity of wild-type Bacillus circulans xylanase (BcX) is set by the pK(a) values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these pK(a) values and thereby shift the pH(opt) at which BcX is optimally active. Altering the global charge of BcX through random succinylation had no significant effect. Mutation of residues near or within the active site of BcX, but not directly contacting the catalytic carboxyls, either had little effect or reduced its pH(opt), primarily by lowering the apparent pK(a) value of Glu78. However, mutations causing the largest pK(a) changes also impaired activity. Although not found as a general acid/base in naturally occurring xylanases, substitution of Glu172 with a His lowered the pH(opt) of BcX from 5.6 to 4.7 while retaining 8% activity toward a xylobioside substrate. Mutation of Asn35, which contacts Glu172, to either His or Glu also led to a reduction in pH(opt) by ~1.2 units. Detailed pK(a) measurements by NMR spectroscopy revealed that, despite the opposite charges of the introduced residues, both the N35H and N35E forms of BcX utilize a reverse protonation mechanism. In this mechanism, the pK(a) value of the general acid is lower than that of the nucleophile, and only a small population of enzyme is in a catalytically competent ionization state. However, overall activity is maintained due to the increased strength of the general acid. This study illustrates several routes for altering the pH-dependent properties of xylanases, while also providing valuable insights into complex protein electrostatics.

The zinc regulated antivirulence pathway of Salmonella is a multiprotein immunoglobulin adhesion system.

The co-evolutionary relationship between pathogen and host has led to a regulatory cycle between virulence factors needed for survival and antivirulence factors required for host transmission. This is exemplified in Salmonella spp. by the zirTS antivirulence genes: a secretion pathway comprised of the outer membrane transporter ZirT, and its secreted partner, ZirS. ZirTS act within the gastrointestinal tract to function as a virulence modulator and during Salmonella shedding in anticipation of a new host. Together, ZirT and ZirS decrease virulence by lowering bacterial colonization at systemic sites through an unknown mechanism. To understand this mechanism, we have probed the zirTS pathway both structurally and biochemically. The NMR derived structural ensemble of the C-terminal domain of ZirS reveals an immunoglobin superfamily fold (IgSF). Stable isotope labeling by amino acids in cell culture experiments show that the ZirS IgSF domain interacts with its transporter ZirT, and reveal a new protein interaction partner of the pathway, a protein encoded adjacent to zirTS that we have designated as ZirU. ZirU is secreted by ZirT and is also a predicted IgSF. Biochemical analysis delineates ZirT into an N-terminal porin-like ß domain and C-terminal extracellular soluble IgSF domain, whereas biophysical characterization suggests that the transporter undergoes self-association in a concentration-dependent manner. We observe that ZirS and ZirU directly interact with each other and with the extracellular domains of ZirT. Here we show that the zir antivirulence pathway is a multiprotein immunoglobulin adhesion system consisting of a complex interplay between ZirS, ZirT, and ZirU.

Protein dielectric constants determined from NMR chemical shift perturbations.

Understanding the connection between protein structure and function requires a quantitative understanding of electrostatic effects. Structure-based electrostatic calculations are essential for this purpose, but their use has been limited by a long-standing discussion on which value to use for the dielectric constants (ε(eff) and ε(p)) required in Coulombic and Poisson-Boltzmann models. The currently used values for ε(eff) and ε(p) are essentially empirical parameters calibrated against thermodynamic properties that are indirect measurements of protein electric fields. We determine optimal values for ε(eff) and ε(p) by measuring protein electric fields in solution using direct detection of NMR chemical shift perturbations (CSPs). We measured CSPs in 14 proteins to get a broad and general characterization of electric fields. Coulomb's law reproduces the measured CSPs optimally with a protein dielectric constant (ε(eff)) from 3 to 13, with an optimal value across all proteins of 6.5. However, when the water-protein interface is treated with finite difference Poisson-Boltzmann calculations, the optimal protein dielectric constant (ε(p)) ranged from 2 to 5 with an optimum of 3. It is striking how similar this value is to the dielectric constant of 2-4 measured for protein powders and how different it is from the ε(p) of 6-20 used in models based on the Poisson-Boltzmann equation when calculating thermodynamic parameters. Because the value of ε(p) = 3 is obtained by analysis of NMR chemical shift perturbations instead of thermodynamic parameters such as pK(a) values, it is likely to describe only the electric field and thus represent a more general, intrinsic, and transferable ε(p) common to most folded proteins.

A pleckstrin homology-related domain in SHIP1 mediates membrane localization during Fcγ receptor-induced phagocytosis.

SH2 domain-containing inositol-5'-phosphatase-1 (SHIP1) inhibits inflammation by hydrolyzing phosphoinositide-3'-kinase generated membrane phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). Bioinformatic analysis of SHIP1 from multiple species revealed a pleckstrin homololgy-related (PH-R) domain, which we hypothesize mediates SHIP1's association with the membrane, a requirement for its biological function. Recombinant murine SHIP1 PH-R domain was subjected to biophysical and biochemical analysis. Residues K370 and K397 were found to be important for PH-R domain association with membrane PIP(3). Wild-type PH-R domain bound PIP(3) with 1.9 ± 0.2 nM affinity, while the affinity of a K370A/K397A substituted mutant was too low to measure. Wild-type (but not the K370A/K397A substituted) full-length SHIP1 protein, reconstitutes normal inhibition of Fcγ receptor-mediated phagocytosis when introduced into SHIP1(-/-) murine macrophages, reducing the number of phagocytic events by 2-fold as compared to SHIP1(-/-) cells. In fact, the PH-R-mediated membrane interaction appears to be a major mechanism by which SHIP1 is recruited to the membrane, since the K370A/K397A substitution reduced the recruitment of both full-length SHIP1 and the PH-R domain by ≥2-fold. We have previously shown that SHIP1 enzyme activity can be targeted for therapeutic purposes. The current studies suggest that molecules targeting the PH-R domain can also modulate SHIP1 function.